11β-HSDl在非酒精性脂肪性肝病大鼠肝脏中的表达及大黄素的干预作用

Expression of 11β-HSDl in liver of rat with non-alcoholic fatty liver disease and the intervention effects of emodin

目的探讨11β-羟基类固醇脱氢酶1(11β-hydroxysteroid dehydrogenase type 1,11β-HSDl)在非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)大鼠肝脏中的表达及大黄素的干预作用。方法将30只雄性Wistar大鼠随机分为对照组、模型组和治疗组,每组10只。对照组给予普通饲料,模型组和治疗组喂以自制高脂饲料,从第9周开始治疗组给予大黄素干预治疗,第14周末实验结束。计算各组大鼠肝指数,检测血液和肝组织生物化学指标,包括丙氨酸氨基转移酶(alaninne aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、总胆固醇(totalcholesterol,TG)、甘油三酯(triglycerides,TC)、高密度脂蛋白(high density lipoprotein,HDL)、游离脂肪酸(free fatty acid,FFA)及血清肿瘤坏死因子-α(tumor necrosis factorα,TNF-α),采用实时荧光定量聚合酶链式反应(real time polymerase chain reaction,RT-PCR)检测肝组织11β-HSD1 mRNA相对表达量,采用Western blot检测肝组织11β-HSD1蛋白相对表达量,于光学显微镜下观察各组大鼠的肝组织病理。结果对照组、模型组和治疗组肝指数分别为(23.4±1.2)mg/g、(25.66±0.99)mg/g、(24.10±1.8)mg/g,差异有统计学意义(F=7.230,P=0.003)。与对照组比较,模型组肝指数显著增高(t=3.725,P<0.001);与模型组比较,治疗组肝指数显著下降(t=2.523,P=0.029)。对照组、模型组和治疗组血液ALT[(42.60±8.78)U/L vs(70.00±11.60)U/L vs(48.90±12.25)U/L]、AST[(99.40±12.99)U/L vs(186.00±20.49)U/L vs(144.7±17.30)U/L]、TC[(1.53±0.17)mmol/L vs(2.11±0.15)mmol/L vs(1.66±0.21)mmol/L]、TG[(0.59±0.06)mmol/L vs(0.68±0.06)mmol/L vs(0.64±0.05)mmol/L]、HDL[(1.05±0.10)mmol/L vs(0.81±0.10)mmol/L vs(0.92±0.09)mmol/L]、FFA[(648±52)μmol/L vs(1081±53)μmol/L vs(764±72)μmol/L]及TNF-α[(0.288±0.068)μg/L vs(0.669±0.051)μg/L vs(0.488±0.054)μg/L]差异均有统计学意义(P均<0.001),与对照组相比,模型组大鼠血液ALT、AST、TC、TG、FFA及TNF-α水平均显著升高(P均<0.05),HDL显著降低(P<0.001);与模型组比较,治疗组上述各项指标均有显著改善(P均<0.05)。对照组、模型组和治疗组大鼠肝脏TC[(1.44±0.12)mmol/g vs(2.10±0.24)mmol/g vs(1.63±0.24)mmol/g]、TG[(4.58±0.41)mmol/g vs(5.73±0.39)mmol/g vs(4.89±0.32)mmol/g]、FFA[(625±95)μmol/g vs(1436±174)μmol/g vs(803±152)μmol/g]及TNF-α[(0.506±0.104)μg/g vs(1.124±0.104)μg/g vs(0.855±0.098)μg/g]差异均有统计学意义(P均<0.001)。与对照组相比,模型组大鼠肝脏有明显脂质沉积,表现为TC、TG、FFA水平显著升高(P均<0.001),且肝组织TNF-α水平也显著升高(P<0.001);与模型组比较,治疗组以上各项指标均显著下降(P均<0.001)。对照组、模型组和治疗组大鼠肝组织11β-HSD1 mRNA(0.052±0.006 vs 0.194±0.026 vs 0.104±0.020)及蛋白(0.112±0.026 vs 0.380±0.096 vs 0.195±0.038)表达差异均有统计学意义(P均<0.001),与对照组比较,模型组肝组织11β-HSD1 mRNA及蛋白表达显著升高(P均<0.001);与模型组比较,治疗组肝组织11β-HSD1 mRNA及蛋白表达均显著降低(P均<0.001)。肝组织病理显示对照组大鼠肝组织细胞结构完整,肝小叶清晰;模型组大鼠肝组织出现明显的肝脂肪变性及大量炎性细胞浸润;与模型组比较,治疗组大鼠肝组织变性和炎性细胞浸润明显减少。结论大黄素可降低NAFLD大鼠肝指数,改善肝功能,降低血脂及肝脏脂质沉积,减轻肝组织病理改变,同时显著抑制肝组织11β-HSD1 mRNA和蛋白表达量,提示大黄素对NAFLD有保护作用,其作用机制可能与抑制11β-HSD1活性有关。

Objective To investigate the expression of 11β-hydroxysteroid dehydrogenase type 1(11β-HSD1)in rats with non-alcoholic fatty liver disease(NAFLD)and the intervention effect of emodin.Methods Total of 30 male Wistar rats were randomly divided into control group,model group and treatment group,10 cases in each group.Rats in control group were fed with normal diet,and rats in model and treatment group were fed with high-fat diet.The treatment group was given emodin from the 9th week,and the experiment was completed at the end of the 14th week.The hepatic index of rats was calculated,and biochemical indexes of blood and liver tissues were detected,including alaninne aminotransferase(ALT),aspartate aminotransferase(AST),totalcholesterol(TG),triglycerides(TC),high density lipoprotein(HDL),free fatty acid(FFA)and tumor necrosis factorα(TNF-α).The relative expression of 11β-HSD1 mRNA in liver tissue was detected by real time polymerase chain reaction(RT-PCR),and the relative expression of 11β-HSD1 protein was detected by Western blot.Liver histopathology was observed under a light microscope.Results The hepatic indexes of rats in control group,model group and treatment group were(23.4±1.2)mg/g,(25.66±0.99)mg/g and(24.10±1.8)mg/g,respectively,the difference was statistically significant(F=7.230,P=0.003).Compared with control group,hepatic index of rats in model group increased significantly(t=3.725,P<0.001).Compared with model group,hepatic index of rats in the treatment group decreased significantly(t=2.523,P=0.029).The differences of serum ALT[(42.60±8.78)U/L vs(70.00±11.60)U/L vs(48.90±12.25)U/L],AST[(99.40±12.99)U/L vs(186.00±20.49)U/L vs(144.7±17.30)U/L],TC[(1.53±0.17)mmol/L vs(2.11±0.15)mmol/L vs(1.66±0.21)mmol/L],TG[(0.59±0.06)mmol/L vs(0.68±0.06)mmol/L vs(0.64±0.05)mmol/L,HDL[(1.05±0.10)mmol/L vs(0.81±0.10)mmol/L vs(0.92±0.09)mmol/L,FFA[(648±52)μmol/L vs(1081±53)μmol/L vs(764±72)μmol/L]and TNF-α[(0.288±0.068)μg/L vs(0.669±0.051)μg/L vs(0.488±0.054)μg/L]of rats in control group,model group and treatment group were statistically significant(all P<0.001).Compared with control group,the serum levels of ALT,AST,TC,TG,FFA and TNF-αof rats in model group increased significantly(all P<0.001)and HDL decreased significantly(P<0.001).Compared with model group,the above indexes in the treatment group were significantly improved(all P<0.05).The differences of liver TC[(1.44±0.12)mmol/g vs(2.10±0.24)mmol/g vs(1.63±0.24)mmol/g],TG[(4.58±0.41)mmol/g vs(5.73±0.39)mmol/g vs(4.89±0.32)mmol/g],FFA[(625±95)μmol/g vs(1436±174)μmol/g vs(803±152)μmol/g]and TNF-α[(0.506±0.104)μg/g vs(1.124±0.104)μg/g vs(0.855±0.098)μg/g]of rats in control group,model group and treatment group were statistically significant(all P<0.001).Compared with control group,there was obvious lipid deposition in liver of rats in model group with increasing levels of TC,TG,FFA and TNF-α(all P<0.001).Compared with model group,all the above indexes of rats in treatment group were significantly decreased(all P<0.001).The differences of 11β-HSD1 mRNA(0.052±0.006 vs 0.194±0.026 vs 0.104±0.020)and protein(0.112±0.026 vs 0.380±0.096 vs 0.195±0.038)in liver tissues of rats in control group,model group and treatment group were statistically significant(all P<0.001).Compared with control group,the expression of 11β-HSD1 mRNA and protein of rats in model group were significantly increased(all P<0.001);compared with model group,the expression of 11β-HSD1 mRNA and protein of rats in treatment group were significantly decreased(all P<0.001).Pathological examination of liver tissue showed that the cell structure of liver tissue was intact and the hepatic lobule was clear in control group.Hepatic steatosis and inflammatory cell infiltration were observed in model group.Compared with model group,liver tissue degeneration and inflammatory cell infiltration were significantly reduced in treatment group.Conclusions Emodin can reduce hepatic index,improve liver function,reduce blood lipid and lipid deposition in liver,alleviate pathological changes of liver tissue,and significantly inhibit the expression of 11β-HSD1 mRNA and protein in liver tissue of rats with NAFLD,suggesting that emodin has a protective effect on NAFLD,and its mechanism may be related to the inhibition of 11β-HSD1 activity.